Non-Invasive Pre-Natal Diagnostics
Non-invasive pre-natal diagnostics are a growing area of development, in part due to increasing parental age (and therefore genetic risk) and the presence of genetic abnormalities in a significant percentage of the infant population. The tests currently on the market, Ariosa, Verify and XX from Verinata, Natera and Sequenom respectively rely on the detection of key abnormalities via counts of molecules derived from different chromosomes in the blood of the pregnant mother, and enable detection of Trisomies 21, 18 and 13.
While these tests have gained relatively widespread market adoption, they are limited to being marketed as a ‘screening’ test due to their relatively low positive predictive value (PPV). As an example, one such screening test, when testing for Trisomy 18, has true positive rate of about 40 patients in 400 positive screening tests. That translates to about a 10% PPV. Because of the potential for false positives, a positive result using the non-invasive test needs to be followed up by an amniocentesis confirmatory test that has a much lower error rate (and higher PPV).
The high false positive rate (and difficulty in developing a true diagnostic) is due to the fact that fetal cell-free DNA (fcfDNA) is present in concentrations of only 4-12% as compared to the mother's cell-free DNA (cfDNA). Consequently, for a fetal trisomy (say Trisomy 18), if there are 3 as opposed to 2 copies of chromosome 18, the change in a sample that is 5% fetal DNA is only (5%/2)=2.5%. The copy number determination therefore needs to be very accurate, as data has a greater than 5% spread and statistical methods need to be employed to determine any imbalance while sequencing at a high depth of coverage. There are a number of different commercial tests based on cfDNA and this type of consideration.
While previous tests have been proposed based on fetal cells in the mother's blood, they have been hard to develop into commercial tests because of the variable cell number recovered and the relatively low concentration of fetal cells to maternal cells in the final sample. A number of different modalities have been used to separate fetal cells out using either posts in a microfluidic channel, macro scale immunomagnetic separation, size (Isolation by Size of Tumor cells i.e. ISET) or ferromagnetic properties of red blood cells. No one modality has demonstrated fetal cell purities of above 10% post enrichment, and thereby low purities have precluded commercial development. For a more thorough treatment of these and related topics, the reader is referred to U.S. Pat. Nos. 8,058,056, 8,293,524 and U.S. Patent Application Publication No. 2013/0017538 A1.
Single Cell Analysis
In areas unrelated to prenatal diagnostics, methods and devices have been proposed to enable the analysis of single cells. The simplest approach, termed Limiting Dilution, consists of measuring the concentration of cells in a certain volume, followed by mixing of the whole sample and dispensing a volume that is expected to contain a single cell in each of many wells. This has the limitation of resulting in 1 cell on average per well, and a distribution of 0, 1, and 2 cells with a few higher numbers, but where only about 60% of wells contain a single cell in optimized protocols. Another related approach is to utilize a fluorescence activated cell sorter (FACS) instrument in order to sort single cells directly into wells of a well plate. This approach suffers from some of the same limitations in success rate, and requires expensive equipment to perform.
A number of different approaches for isolating and analyzing single cells using microfluidic devices have also been proposed. One approach previously commercialized by Fluidigm, Inc. as part of their C1 product offering utilizes a trap within the flow of a microfluidic channel, and microchannel based valve-ing to isolate the cell and to perform lysis, followed by nucleic acid amplification and extraction for up to 96 cells.
One approach previously proposed for single cell immobilization was to use an array of lateral junctions. The approach provided for a method of either patch clamp recording or electroporation of single cells, which would lead to lysis, but did not clearly describe a method for further analyzing the resulting nucleic acids from single cells by performing the necessary amplification, sequestration and extraction of the amplified material. For a more rigorous treatment of this approach, the reader is referred to U.S. Pat. No. 8,058,056. The single cell immobilization methods described may also be followed up by microscopy based detection of chromosome abnormalities for each cell.